Over the past year and a half, we have developed a new interferometric technique in fluorescent imaging called spectral self-interference fluorescent microscopy (SFM). The technique utilizes the spectral oscillations emitted by a fluorophore located a distance of several wavelengths above a reflecting surface. These spectral oscillations are due to the self-interference from the direct and reflected emitted light, analysis of which yields the vertical position of the fluorophore to within a few nanometers. This proposal is to demonstrate SFM as a nanometer resolution fluorescent microscopy technique in both artificial and biological model systems, apply its unique resolution capability to novel biological questions in vivo, and finally, extend the capabilities to arbitrary sectioning and eventual 3D, real-time nanoscale fluorescent imaging. The overall, long-term goal is to develop, demonstrate and apply in vivo subcellular microscopy at an ultimate resolution of 10 nanometers. The precise three-dimensional localization of proteins within prokaryotic cells is key to many cellular functions, including cell cycle, DNA replication, development, motility, and adhesion. As yet, the basic mechanisms that mediate three-dimensional targeting of proteins in prokaryotes remain largely unknown. Dr. MB Goldberg's laboratory has shown that the targeting of the Shigella actin assembly protein IcsA to the bacterial old pole occurs in the bacterial cytoplasm and involves two specific regions of the polypeptide. We will apply the SFM developed in this proposal to analyze with sub-cellular resolution in live bacteria specific interactions of IcsA with proteins involved in its secretion.